石斑鱼人工育苗技术研究

通过对点带石斑鱼人工育苗生产试验,从饵料准备、环境条件控制、管理措施等方面进行了探索。共培育出规格5～8cm的石斑鱼苗60多万尾。     

光周期对点带石斑鱼幼鱼生长及肝脏溶菌酶活性的影响

[目的]研究不同光周期对点带石斑鱼幼鱼生长、摄食及肝脏溶菌酶活性的影响。[方法]试验设计持续光照组、对照组、持续黑暗组,每组3个平行,计算生长率,测定肝脏溶菌酶活性,并作比较。[结果]持续光照组与其他2组相比,相对增重率与溶菌酶活性均最高,相对增重率为对照组的107.3%,溶菌酶活性为对照组的103.4%;对照组与持续光照组差异不显著;持续黑暗组显著低于对照组。[结论]点带石斑鱼幼鱼的生长、免疫及行为与光周期有紧密联系。     

斜带石斑鱼肝细胞分离及原代培养方法的建立

本研究以斜带石斑鱼肝细胞为实验对象,在不同培养条件下进行原代培养,旨在探讨稳定可靠的斜带石斑鱼肝细胞分离及原代培养方法。采用组织块分离法和胰蛋白酶(含EDTA)消化法分离肝细胞,并通过密度梯度离心法分离纯化肝细胞,细胞悬液于DMEM/F-12、M199和L-15培养液中培养;细胞活力及数量采用血球计数板计数,并通过MTT法测定细胞增殖率;同时,测定不同时间培养上清液中乳酸脱氢酶(LDH)活性、白蛋白(ALB)和尿素氮(BUN)的含量,以分析肝细胞生长状态。结果表明,组织块方法不适于斜带石斑鱼肝细胞的培养,未见细胞从组织块中迁出,而胰蛋白酶消化法获得良好稳定的培养效果,细胞产量达到1.6×108个/g肝重,活细胞数达到95%;L-15培养基细胞生长明显优于DMEM/F-12和M199培养基;启动原代培养的48~72 h阶段肝细胞生长代谢旺盛,培养上清液中LDH活性显著降低,ALB和BUN含量显著升高。结果显示,0.25%的胰蛋白酶常温消化法适合斜带石斑鱼肝细胞的分离,斜带石斑鱼肝细胞原代培养的最适培养基为L-15培养基,肝细胞在启动原代培养的48~72 h生长代谢旺盛。     

Using Bacillus subtilis E20‐fermented soybean meal as replacement for fish meal in the diet of orange‐spotted grouper (Epinephelus coioides,Hamilton)

The potential of Bacillus subtilis E20‐fermented soybean meal (FSBM) as a partial alternative component of fish meal (FM) in fed diets of orange‐spotted grouper (Epinephelus coioides) was evaluated in this study. An FM‐based diet and seven diets containing 10%, 20% and 30% and 10%, 20%, 30% and 40% of FM replaced by soybean meal (SBM) and FSBM, respectively, were fed to grouper for 84 days to evaluate possible substitution levels of FM by tracking growth performance, digestive enzyme activity and morphological changes in the liver and distal intestine. No significant differences in survival and muscle composition of grouper were found between controls and treatments. Growth performance and feed efficiency of fish fed diets with FM replaced by FSBM up to 30% were not significantly different from controls, whereas significantly decreased growth performance and feed efficiency occurred with diets containing >20% of SBM. Based on the feed efficiency, the maximum substituted levels of FM by SBM and FSBM in grouper diets were 18.36% and 29.32%, respectively, based on broken‐line analyses. Histopathological changes in the liver and distal intestine, and significantly lower activity levels of digestive enzymes, including pepsin in the stomach and trypsin, chymotrypsin, amylase and lipase in the distal intestine, were found in fish fed a diet containing 30% of FM replaced by SBM. However, these parameters were improved by the substitution of FSBM. It is therefore believed that FSBM has great potential to be used as a protein source in grouper diets in partial replacement of FM.     

不同维生素E水平饲料对云纹石斑鱼幼鱼低盐胁迫前后抗氧化和渗透压调节功能的比较

维生素E(VE)在鱼类繁殖、生长代谢、抗氧化能力和免疫机能等方面有至关重要的作用。为探究饲料VE添加量对云纹石斑鱼低盐度胁迫下抗氧化和渗透压调节功能的作用,本实验设计0、20、40、80和160 mg/kg等5个VE水平饵料添加量,依次为A、B、C、D、E组,饲喂云纹石斑鱼幼鱼56 d。然后进行6 h低盐度(12)胁迫实验,测试皮质醇(COR)、葡萄糖(GLU)、乳酸(LD)等血清生化指标,超氧化物岐化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、总抗氧化能力(T-AOC)和丙二醛(MDA)等抗氧化指标,以及鳃Na⁺/K⁺-ATP酶(NKA)、Ca²⁺-ATP酶活力等。结果显示,血清COR含量最高为盐度胁迫后A(32.74±1.53) ng/mL,最低为胁迫前E(19.06±3.88) ng/mL;GLU含量最高为盐度胁迫后A(16.46±0.99) mmol/L,最低为养殖前O(7.90±0.34) mmol/L;LD含量最高为低盐度胁迫前C(1.94±0.15) mmol/L,最低为胁迫后E组(1.30±0.06) mmol/L。说明VE的添加可以增强云纹石斑鱼抗应激的能力,且在47.524~134.566 mg/kg(B~D)的添加范围,效果较好。血清SOD、CAT、GSH-Px活力、T-AOC和MDA含量与投喂饲料中VE水平密切相关,SOD最高为胁迫前C(105.29±9.07) U/mL,最低为胁迫后A(11.23±2.30) U/mL;CAT最高为养殖前O(4.09±0.17) U/mL,最低为胁迫后A(0.35±0.10) U/mL;GSH-Px最高为胁迫前E(972.58±55.35) U,最低为胁迫后A(47.90±10.64) U;MDA最低为胁迫前D(33.48±2.34) nmol/mL,最高为胁迫后A(101.79±7.79) nmol/mL。饲料中添加91.378~178.924 mg/kg(C~E)水平VE对其抗氧化能力有增强作用。VE可增强NKA活力,当VE添加量为91.378 mg/kg(C)时,低盐度胁迫对NKA影响较小。而添加91.378~178.924 mg/kg(C~E)水平VE可增强云纹石斑鱼Ca²⁺-ATP酶活力。研究表明,饲料中添加91.378~134.566 mg/kg范围的维生素E,可以增强云纹石斑鱼的机体活力、抗应激及环境胁迫能力。     

外源人绒毛膜促性腺激素诱导黑边石斑鱼排卵的研究

【目的】了解外源人绒毛膜促性腺激素(human chorionic gonadotropin,HCG)诱导黑边石斑鱼(Epinephelus fasciatus)排卵的影响。【方法】以成熟的黑边石斑鱼为研究对象,测量其体长、体质量和肥满度后,在2018年7—8月,使用HCG激素(剂量为200 IU/kg或500 IU/kg)注射分别对6尾雌鱼进行成熟诱导。【结果】在200 IU/kg HCG处理中,注射24 h前有5尾雌鱼的卵母细胞处于第三次卵黄球前期阶段;注射48 h后2尾雌鱼出现核移动卵母细胞,1尾雌鱼的卵母细胞为第三次卵黄球后期阶段;注射60 h后1尾雌鱼出现排卵现象,其余5尾雌鱼均为第三次卵黄球后期阶段。在200 IU/kg HCG处理的排卵个体中,注射时其卵母细胞直径为477.0 μm,注射后60 h卵母细胞的直径增加至624.4 μm。此外,经催产、排卵、受精和孵化后共获得总卵数16 906粒,受精率为68.7%,孵化率为43.0%。而500 IU/kg HCG处理的黑边石斑鱼排卵失败。【结论】使用剂量为200 IU/kg的外源HCG激素可诱导黑边石斑鱼成熟且排卵,但为了提高排卵率、受精率和孵化率,其注射剂量和效应时间还需要进一步调节。     

用微卫星标记研究南海四种石斑鱼的遗传变异与种间关系(英文)

[目的]从DNA分子水平揭示我国南海的四种重要经济海水鱼类点带石斑鱼(Epinephelusmalabaricus)、斜带石斑鱼(E.coioides)、鲑点石斑鱼(E.fario)、蜂巢石斑鱼(E.merra)的遗传群体变异水平与种间关系。[方法]本研究利用从GenBank中搜索到的21对微卫星引物,对点带石斑鱼、斜带石斑鱼、鲑点石斑鱼及蜂巢石斑鱼的等位基因数A、有效等位基因数Ne、多态信息含量PIC、观察遗传杂合度Ho、期望遗传杂合度He等遗传参数进行检测,并采用UPGMA类聚分析法探讨这四种石斑鱼的种间关系。[结果]点带石斑鱼群体的A、Ne、PIC、Ho和He等遗传参数的平均值分别为4.38±1.60、3.69±0.86、0.69±0.08、0.67±0.08、0.72±0.06,斜带石斑鱼群体的为3.88±1.13、3.55±1.04、0.66±0.10、0.68±0.21、0.70±0.08,鲑点石斑鱼群体的为6.00±1.07、4.68±0.65、0.78±0.03、0.73±0.25、0.79±0.03,蜂巢石斑鱼群体的为5.50±1.07、4.58±0.80、0.76±0.05、0.75±0.18、0.78±0.04。[结论]综合比较上述数值可知,四个石斑鱼群体的遗传变异程度依次为鲑点石斑鱼群体>蜂巢石斑鱼群体>点带石斑鱼>斜带石斑鱼群体;与其他海水鱼类的遗传变异水平比较,四种石斑鱼的群体遗传变异水平都较高,说明我国这些石斑鱼种类的遗传种质资源尚好。另外,遗传距离与类聚分析表明,点带石斑鱼与斜带石斑鱼遗传距离最近,而斜带石斑鱼与蜂巢石斑鱼的亲缘关系最远。     

赤点石斑鱼神经坏死病毒MCP基因原核表达条件优化(英文)

[Objective] To optimize the prokaryotic expression of MCP gene of red-spotted grouper nervous necrosis virus. [Method] The MCP gene was amplified from red-spotted grouper nervous necrosis viral genome by RT-PCR. The recombinant expression vector pRSET A-MCP was constructed and transformed into BL21(DE3)plysS to express proteins with induction in different media, at different pH, or at different temperatures. [Result] The expression level of recombinant bacteria reached a peak with induction under the following condition: SOB or LB medium, pH 7.0, 37 ℃ while the fusion protein was about 44.5 kD in molecular weight. [Conclusion] This study provided a basis for the development of RGNNV-MCP vaccine.     

饲料中大豆卵磷脂/鱼油比例对斜带石斑鱼稚鱼生长发育及肠道组织的影响

为探讨饲料中不同大豆卵磷脂/鱼油比例对斜带石斑鱼稚鱼成活率、生长行为、饲料利用以及肠道组织学的影响,本实验设计了4种添加不同大豆卵磷脂/鱼油比率的微颗粒饲料：0%/22%（饲料1）,5%/14%（饲料2）,10%/8%（饲料3）以及15%/7%（饲料4）,饲喂斜带石斑鱼稚鱼。每种实验饲料均设3个平行。试验持续40d。结果表明,饲料中不同大豆卵磷脂/鱼油比例对斜带石斑鱼稚鱼生长以及成活率有显著影响（P≤0.05）。饲料3和饲料4的增重分别为：（449.6±11.4）g和（588.3±61.7）g,显著高于饲料1的数值（185.0±2.5）（P≤0.05）。饲料1鱼体脂肪含量为：（154.7±12.8）g·kg-1,显著低于其他饲料组（P≤0.05）,但其鱼体蛋白含量为（728.3±25.5）g·kg-1,显著高于其他饲料组。饲料效率、肥满度、特定生长率、摄食量和蛋白质效率以及终末体重中,4种饲料之间无显著性差异。随饲料卵磷脂/鱼油比例升高,石斑鱼稚鱼肠道脂滴生成数量出现下降,且在饲料4中未观察到肠道脂滴。研究表明斜带石斑鱼稚鱼生长发育阶段需要较高的饲料卵磷脂/鱼油比例（如：15%/7%）。     

Effects of dietary live or heat‐inactivated autochthonous Bacillus pumilus SE5 on growth performance,immune responses and immune gene expression in grouper Epinephelus coioides

To evaluate the possible dietary application of live and heat‐inactivated probiotic Bacillus pumilus SE5 in grouper Epinephelus coioides, juveniles (14.6 ± 0.2 g) were fed either a basal control diet (without probiotic) or the basal diet supplemented with 1.0 × 10⁸ CFU g^?1 live (T1) and heat‐inactivated B. pumilus SE5 (T2). The heat‐inactivated probiotic significantly improved the final weight, weight gain (WG) and specific growth rate (SGR) at day 60 and significantly decreased the feed conversion ratio (FCR) at day 30 and 60, while the viable probiotic significantly decreased the FCR at day 60 (P < 0.05). Phagocytic activity, serum complement C3 and IgM levels as well as SOD activity elevated significantly in fish fed the heat‐inactivated probiotic for 60 days (P < 0.05). Furthermore, the heat‐inactivated probiotic remarkably up‐regulated expression of TLR2 and pro‐inflammatory cytokines (IL‐8 and IL‐1β) in head kidney (P < 0.05), but the viable probiotic failed to do so. These results indicated that heat‐inactivated B. pumilus SE5 can effectively improve the growth performance and immune responses of E. coioides.